Project Summary Cystic fibrosis (CF) is a genetically inherited disease that is due to loss-of-function mutations in the CFTR gene. Thousands of mutations in CFTR have been identified and much of the efforts for CF therapy have focused on rescuing the dysfunction of the CFTR mutations. However, developing therapies for the thousands of mutations that are thought to cause to CF would be challenging. Others have therefore sought to activate alternative sources of anion channels in the lungs, with the expectation that these therapies would not depend on the specific CFTR mutation. In the past decade, the identification of one such candidate, the calcium-activated chloride channel, TMEM16A, has prompted some groups to propose activation of this channel to restore anion currents in CF airway. Our group has found that CLCA1, a specific and direct regulator of TMEM16A, can increase chloride current density by preventing removal of the channel from the plasma membrane. The 18 kDa vWA domain within CLCA1 is able to carry out this activity alone, in HEK293T cells and in primary CF airway cells. These findings raise the question of how a fully secreted regulator is able to influence the intracellular trafficking of a channel. Furthermore, it not known whether the vWA domain of CLCA1 can restore mucous properties in CF airway epithelium. To address these questions, I will study the cellular mechanism by which the CLCA1 vWA domain affects trafficking of TMEM16A using imaging methods and mass spectrometry. Using X-ray crystallography, NMR and biophysical binding studies, I will identify the key molecular contacts that govern the interaction of these proteins. Finally, I will apply the vWA domain to differentiated airway culture derived from primary CF airway cells to study its effects on TMEM16A trafficking, epithelial anion secretion and epithelial mucous properties. The ultimate goal of this project is to understand the mechanism of the CLCA1-TMEM16A interaction as a model for developing universal therapy for cystic fibrosis.